First report of Botrytis cinerea causing gray mold on Prunella vulgaris in Hubei province, China.

Abstract

Prunella vulgaris L. is a perennial herb plant of the Lamiaceae family, and its dried spicas have been widely used as medicine, health-promoting food or tea around the world. P. vulgaris is distributed all over the world, such as Europe, Asia, northwestern Africa and North America, as well as the Huaihe River Basin and the middle and lower Yangtze River Basin in China. In February 2022, a serious disease like gray mold occurred in planting fields of P. vulgaris in Wuhan, Hubei (N30°27'07″, E114°15'49″), causing approximately 20% of plants were diseased in the field. Early symptoms were characterized by small, round gray-brown lesions on the leaves of P. vulgaris. Later, a large number of stems and leaves are wilted or necrotic, associated with wet rot and waterlogged spots and covered with light gray or grayish white flocculent mildew layer. To determine the causal agent of disease, 10 plants with the typical symptoms were collected from fields. The stems and leaves of diseased plants were cut into pieces (2 to 3 mm×5 mm), disinfected with 75% ethanol for 3 minutes, rinsed 3 times with sterile water. Each lesion sample was isolated and purified using separate PDA petri dishes at 25°C, and ultimately all samples yielded morphologically consistent pure strain colonies. From the 10 isolates obtained, XKC-1 was chosen as a representative isolate for further study. XKC-1 colonies showed gray aerial mycelia, which were fast-growing and grew over the whole plate (9 cm) after 4 days. In addition, some black and hard sclerotia (1.88±0.94 mm, n=50) with round or irregular shape developed on the colonies after approximately 10 days of incubation at 25°C (Fig. 2A, B). XKC-1 showed branched conidiophores with enlarged apical cells and numerous conidia (Fig. 2C). Unicellular conidia were colorless or gray, ellipsoid or ovoid, smooth and 7.91-12.38 μm × 10.08-13.82 μm (n=30) in size (Fig. 2D). Based on morphological characteristics, the isolate was initially identified as Botrytis sp. (Ellis 1971). To further identify the species, the genomic DNA of XKC-1 was extracted, and the ITS, LSU and G3PDH genes were amplified with the primers ITS1/ITS4, LROR/LR5 (Zhou et al. 2022) and G3PDH-F/G3PDH-R (Jin et al. 2022), respectively. The results indicated that the ITS (ON090404), LSU (ON090417), and G3PDH (ON169893) sequences were 99.80%, 100% and 99.46% identical to the sequences of Botrytis cinerea Pers. strain (MK370693.1, MN148533.1, MN630267.1), respectively. A phylogenetic tree constructed based on a concatenated sequence (ITS, LSU, G3PDH) using the neighbor-joining method in MEGA7 (Tamura et al. 2013) revealed that XKC-1 grouped with concatenated sequences from three representative B. cinerea isolates in GenBank. Based on the morphological characteristics and molecular identification, the strain XKC-1 was identified as Botrytis cinerea. For pathogenicity tests on detached leaves, 5 mm PDA cakes prepared from XKC-1 were placed on the leaves obtained from healthy P. vulgaris after wounding with a needle (n=10), while PDA medium without mycelia were used as control (25 ± 2°C) (Li et al.2020). Mycelia began to germinate and infect plant tissues at 1 dpi. A large part of the leaves showed water soaked spots covered with mycelia on the surface at 4 dpi. For whole plant inoculations, stem bases of five P. vulgaris seedlings were pierced with sterile needle, and then inoculated with three XKC-1 mycelium PDA cakes. Five plants were inoculated with three PDA cakes without mycelia as a control. After 2-4 days, lesions appeared on the leaves and covered with a gray-white mycelial layer, similar to those observed in the field. However, controls remained symptom free. The pathogen was reisolated from the diseased tissues, the colonies, microscopic characteristics and molecular identification were consistent with those of XKC-1. To our knowledge, this is a first report of B. cinerea causing gray mold on P. vulgaris in Hubei, China. This report would provide resources and reference for controlling of the increased incidence and economic losses of gray mold on P. vulgaris.